Study Protocol Clinical Trial Medicine ® Efficacy and safety clinical trial with efavirenz in patients diagnosed with adult Niemann-pick type C with cognitive impairment Jordi Gascón-Bayarri, MDa,b,c,d, Petru Cristian Simon, MDe, Roser Llop, MD, PhDe, Thiago Carnaval, MDf,* , María Dolores Ledesma, PhDg, Imma Rico, BAa,c, Cristina Sánchez-Castañeda, PhDa,c,h, Jaume Campdelacreu-Fumadó, MD, PhDa,c, Nahum Calvo-Malvar, MDc,i, Mònica Cos, MDc,i, Eugenia de Lama, MDc,i, Montserrat Cortés-Romera, MDc,j, Laura Rodríguez-Bel, MDc,j, Celia Pérez-Sousa, MDk, María Cerdán Sánchez, MDl, Nuria Muelas, MDm, María Dolores Sevillano, MDn, Pablo Mir, MD, PhDo,p,q, Adolfo López de Munain, MD, PhDp,r,s,t, Anna Ferrer, PharmDu, Sebastián Videla, MD, PhDe,f,v Abstract Background: Niemann-Pick disease Type C (NPC) is a genetic, incurable, neurodegenerative disorder. This orphan disease is most frequently caused by mutations in the NPC1 protein, resulting in intralysossomal cholesterol accumulation. NPC1 is found in neuronal cell bodies, axon terminals and synaptosomes, suggesting it plays a role in lysosomal degradation pathway and in synaptic transmission. Neuronal function is especially vulnerable to NPC1 deficiency and synaptic changes seem a key element in disease development. Currently, Miglustat (Zavesca®) is the only approved treatment for NPC. However, preclinical evidence showed that low-dose Efavirenz reverted synaptic defects through pharmacological activation of the enzyme CYP46. Methods: This is a single-center, phase II clinical trial to evaluate the efficacy and safety of Efavirenz in addition to standard of care in patients diagnosed with adult or late juvenile-onset NPC with cognitive impairment. All enrolled patients will be treated orally with 25 mg/d of Efavirenz for 52 weeks (1 year). Secondary objectives include evaluating clinical (neurological and neuropsychological questionnaires) and biological (imaging and biochemical biomarkers) parameters. Discussion: NPC is still an unmet medical need. Although different therapeutic approaches are under study, this is the first clinical trial (to the best of our knowledge) studying the effects of Efavirenz in adult- and late-juvenile-onset NPC. Despite the small sample size and the single-arm design, we expect the results to show Efavirenz’s capacity of activating the CYP46 enzyme to compensate for NPC1 deficiency and correct synaptic changes, therefore compensating cognitive and psychiatric changes in these patients. This study may provide direct benefit to enrolled patients in terms of slowing down the disease progression. Abbreviations: AE = adverse events, BUH = Bellvitge university hospital, CDR-SoB = clinical dementia rating scale-sum of boxes, e-CRF = electronic case report form, EFV = efavirenz, LTP = long-term potentiation, NPC = Niemann-pick disease type C, PI = principal investigator, SAE = serious adverse events, SmPC = summary of product characteristics, SoC = standard of care, STROOP = Stroop color and word test, WAIS = Wechsler adult intelligence scale. Keywords: cognitive impairment, efavirenz, Niemann-pick disease type C, NPC1 1. Introduction years after diagnosis. From a clinical perspective, NPC usually affects the viscera (splenomegaly and/or hepatomegaly), but it 1.1. Background and rationale is, above all, a neurological disease. It is a progressive disease Niemann-Pick disease Type C (NPC) is a genetic, neurodegener- without curative treatment and with only 1 treatment approved ative disorder with a life expectancy ranging between 1 – and 30 to date – with limited efficacy (Miglustat, Zavesca®). In 2019, This study received a grant from the Spanish Niemann-Pick Foundation that shall Biomedical Research Institute (IDIBELL), L’Hospitalet DE Llobregat, Barcelona, Spain, cover the Spanish Medicines and Medical Devices Agency fees, insurance policy, d Department of Morphological Sciences, School of Medicine, Autonomous University study medication, additional brain 18FDG PET – scans, electronic Case Report of Barcelona, Bellaterra, Spain, e Clinical Pharmacology Department, Bellvitge Form development, database management, statistical analysis, study coordinator University Hospital, L’Hospitalet de Llobregat, Barcelona, Spain, f Pharmacology fees, monitoring activities, final report, and publication costs. Unit, Department of Pathology and Experimental Therapeutics, School of Medicine and Health Sciences, IDIBELL, University of Barcelona, L’Hospitalet DE Llobregat, The authors have no conflicts of interest to disclose. Barcelona, Spain, g Severo Ochoa Molecular Biology Center (CSIC-UAM), Madrid, The datasets generated during and/or analyzed during the current study are not Spain, h Department of Clinical Psychology and Psychobiology, Neurosciences publicly available, but are available from the corresponding author on reasonable Institute, University of Barcelona, Barcelona, Spain, i Neuroradiology Department, request. Bellvitge University Hospital, L’Hospitalet DE Llobregat, Spain, j Nuclear Medicine- Trial registration: EudraCT Number: 2019-004498-18. PET/CT Department (IDI). Bellvitge University Hospital, L’Hospitalet DE Llobregat, Spain, k Neurology Department, A Coruña University Hospital Complex, A Coruña, Supplemental Digital Content is available for this article. Spain, l Neurology Department, Santa Lucía-Santa María Rosell University Hospital, a Neurology Department, Bellvitge University Hospital, L’Hospitalet DE Llobregat, Cartagena, Spain, m Neurology Department, La Fe University and Polytechnical Barcelona, Spain, b Adult Lysosomal Diseases Clinical Expertise Unit, Bellvitge Hospital, Valencia, Spain, n Neurology Department, Salamanca University University Hospital, L’Hospitalet DE Llobregat, Barcelona, Spain, c Bellvitge Hospital, Salamanca, Spain, o Movement Disorders Unit, Neurology and Clinical 1 Gascón-Bayarri et al. • Medicine (2022) 101:48Medicine adult-onset NPC prevalence in Spain was 27 patients, of which are the rationale for conducting this clinical trial on this orphan ten are being treated at Bellvitge University Hospital (BUH). disease. The disease is classified according to the age of onset of the According to the summary of product characteristics neurological symptoms in: early infantile (before 2 years of (SmPC), EFV was authorized in May 1999 as a non-nucle- age); late infantile (between 3 and 5 years of age); early juvenile oside reverse transcriptase antiretroviral inhibitor and, cur- (between 5 and 14 years of age); late juvenile (between 14 and rently, it is a generic drug used in the combined antiviral 16 years of age); and adult (from 16 years of age onwards). treatment of human immunodeficiency virus-1[13] in infected The clinical presentation includes psychomotor development adults, adolescents and children aged 3 months and older that regression, epilepsy, ataxia, vertical supranuclear ophthalmo- weigh at least 3.5 kg. In adults, the recommended oral dose of plegia, bulbar syndrome, dystonia, splenomegaly, cognitive EFV combined with nucleoside reverse transcriptase inhibi- impairment, behavioral disorders, and symptoms of psychiatric tors with or without a protease inhibitor is 600 mg once a day disorders, with behavioral/cognitive symptoms being most com- (600 mg/d). It is a treatment administered “for life” in patients mon in adults. Altogether, these symptoms point towards synap- infected with HIV. tic changes as a key element in disease development, similar to This clinical trial aims to evaluate the efficacy and safety of other dementias (e.g., Alzheimer’s), where cognitive impairment EFV at an oral dose of 25 mg/d in patients diagnosed with adult correlates with the loss of synapses. or late juvenile-onset NPC with cognitive impairment. In 95% of the cases, genetic mutations in the NPC1 protein are responsible for disease development. The NPC1 protein is involved in the intracellular transport of cholesterol.[1–3] From 1.2. Explanation for the choice of comparators a cellular and molecular perspective, one of the main hallmarks Given the clinical trial characteristics, all patients included in of NPC is the accumulation of cholesterol and sphingolipids in the clinical trial will be treated orally with 25 mg/d of EFV for lysosomal compartments, where NPC1 is found.[4] Although 52 weeks (1 year), in addition to Standard of Care (SoC). lipid accumulation occurs in all cell types, neurons are especially vulnerable to NPC1 deficiency, suggesting that neuronal func- tion is especially related to this protein. Also, NPC1 is found not 1.3. Objectives only in cell bodies, where endosomes and lysosomes are highly abundant,[5] but also in axon terminals and synaptosomes,[6,7] The primary, secondary, and safety objectives are summarized pointing towards a synaptic role from NPC1 other than partici- in Table 1. pating in the lysosomal degradation pathway. Some authors demonstrated the importance of cholesterol synaptic redistribution and elimination for the correct develop- 1.4. Trial design ment of long-term potentiation (LTP).[8] LTP is a major event for This is a single-center, phase II clinical trial to evaluate the effi- synaptic plasticity, involved in processes like memory, learning, cacy and safety of EFV, in addition to SoC, in patients diag- and emotional responses. Cholesterol loss during LTP has been nosed with adult or late juvenile-onset NPC with cognitive associated with the enzyme CYP46A1, mobilized to the plas- impairment. matic membrane in response to synaptic stimulation, hydrox- All enrolled patients will be treated orally with 25 mg/d of ylating cholesterol, making it more soluble, and mediating the EFV for 52 weeks (1 year), in addition to SoC. According to the main pathway for its degradation in the brain.[9,10] SmPC, the recommended dose of EFV as an antiretroviral for Given that NPC1 can bind cholesterol and participate in adults is 600 mg/d orally. intracellular cargo mobilization, we hypothesized that NPC1 would have a synapse-specific role in redistributing cholesterol and mobilizing the CYP46A1 enzyme to the synaptic mem- 2. Methods: Participants, interventions, and brane, required for LTP. We believe that the pharmacological activation of CYP46A1 could compensate for NPC1 deficiency outcomes and correct synaptic changes, therefore compensating cognitive 2.1. Study setting and psychiatric alterations in patients with NPC. Preclinical data on Npcnmf164 mice[11] (which have the D1005G This is a single-center clinical trial. Therefore, this study will mutation in the NPC1 gene and develop NPC slower than be carried out at BUH (L’Hospitalet de Llobregat, Barcelona, NPC1−/− mice) have provided evidence that low-dose Efavirenz Spain) and will be coordinated by the Neurology Department. (EFV) (compared to the dosages used with antiretrovirals) mod- This is a fully equipped tertiary hospital. ifies the morphological, molecular, and functional changes in their synapses.[12] It also improved cognitive and psychiatric changes, reverted synaptic defects through pharmacological 2.2. Eligibility criteria activation of the enzyme CYP46, and increased the life expec- Patients that are to be enrolled in the trial must meet all the tancy of Npcnmf164 mice by 30%.[12] These encouraging results inclusion criteria and none of the exclusion criteria. Neurophysiology Department, Seville Biomedical Institute, Virgen del Rocío Copyright © 2022 the Author(s). Published by Wolters Kluwer Health, Inc. University Hospital/CSIC/University of Seville, Seville, Spain, p Neurodegenerative This is an open access article distributed under the Creative Commons Diseases Network of Biomedical Research Centers (CIBERNED), Madrid, Spain, Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, q Medicine Departament, School of Medicine, University of Seville, Seville, Spain, and reproduction in any medium, provided the original work is properly cited. r Neurology Department, Donostia-Osakidetza University Hospital, San Sebastian, How to cite this article: Gascón-Bayarri J, Simon PC, Llop R, Carnaval T, Spain, s Neuroscience Department, Biodonostia Institute, San Sebastian, Spain, t Ledesma MD, Rico I, Sánchez-Castañeda C, Campdelacreu-Fumadó J, Calvo- Neuroscience Department, University of the Basque Country, San Sebastian, Spain, Malvar N, Cos M, de Lama E, Cortés-Romera M, Rodríguez-Bel L, Pérez- u Pharmacy Department, Bellvitge University Hospital, L’Hospitalet de Llobregat, Sousa C, Cerdán Sánchez M, Muelas N, Sevillano MD, Mir P, López de Munain Barcelona, Spain, v Clinical Research Support Unit (HUB-IDIBELL: Bellvitge A, Ferrer A, Videla S. Efficacy and safety clinical trial with efavirenz in patients University Hospital & Bellvitge Biomedical Research Institute), Clinical Pharmacology diagnosed with adult Niemann-pick type C with cognitive impairment. Medicine Department, Bellvitge University Hospital, L’Hospitalet de Llobregat, Barcelona, Spain. 2022;101:48(e31471). * Correspondence: Thiago Carnaval, Pharmacology Unit, Department of Pathology Received: 8 August 2022 / Accepted: 28 September 2022 and Experimental Therapeutics, School of Medicine and Health Sciences, IDIBELL, University of Barcelona, L’Hospitalet DE Llobregat, Barcelona, Spain. http://dx.doi.org/10.1097/MD.0000000000031471 2 Gascón-Bayarri et al. • Medicine (2022) 101:48www.md-journal.com Table 1 Summarized study objectives. Primary Objective •To evaluate the efficacy* and safety of EFV at an oral dose of 25 mg/d, in addition to standard treatment, in patients diagnosed with adult or late juvenile-onset NPC with cognitive impairment. *Note: Given that adult or late juvenile-onset NPC is rare and causes progressive cognitive impairment, treatment with EFV will be considered effective if it “slows down or stops cognitive impairment in patients diagnosed with adult or late juvenile-onset NPC with cognitive impairment.” Meaning, it is hoped that after 1 year of treatment, these patients remain at the baseline with respect to cognitive impairment. Secondary objectives Clinical • Neurological: To evaluate the change in the following questionnaires/scales:  SARA  EAT-10 score.  PDS • Neuropsychological (protocol used in clinical practice): To evaluate the change in the following questionnaires/ scales:  CDR-SoB  Verbal memory: FCSRT  a-BT  Verbal fluency: Semantic and phonetic cues  TMT A and B  STROOP  WAIS and the block design, symbol search and vocabulary subtests (WAIS-III/WAIS-IV)  BNT, and the Naming and Semantic Knowledge subtests  JLO  DEX  NPI  AES  BDI  C-SSRS Biological • To evaluate the change in functional and imaging biomarkers related to NPC:  18 FDG PET—scan  Brain MRI  Abdominal ultrasound  Oculography • Evaluate the change in biochemical biomarkers related to NPC:  Oxysterols  Lyso-SM-509  24-OH-Cholesterol  Beta-Amyloid, Tau, and phosphorylated Tau Safety • To evaluate the safety of EFV in patients diagnosed with adult or late juvenile-onset NPC with cognitive impairment. 18 FDG PET—scan = 18Fluorodeoxyglucose Positron emission tomography scan, a-BT = abbreviated Barcelona test, AES = apathy evaluation scale, BDI = Beck’s depression inventory, BNT = Boston naming test, CDR-SoB = clinical dementia rating scale-sum of boxes, C-SSRS = Columbia suicide severity rating scale, DEX = dysexecutive questionnaire, EAT-10 = the eating assessment tool score, EFV = efavirenz, FCSRT = free and cued selective reminding test, JLO = judgement of line orientation test, MRI = magnetic resonance imaging, NPC = Niemann-Pick disease type C, NPI = neuropsychiatric inventory, PDS = Pineda disability scale, SARA = scale for the assessment and rating of ataxia, STROOP = stroop color and word test, TMT = trail making test, WAIS = Wechsler adult intelligence scale. 2.2.1. Inclusion criteria. Patients must: • Be on Miglustat treatment (only treatment indicated for NPC) with a stable dose for the last 3 months. • Be at least 14 years of age, of both genders. • In the case of women of child-bearing potential (i.e., the • Be diagnosed with adult-onset NPC based on a genetic period between menarche and menopause), have a negative study with 2 genetic mutations related to this disorder OR pregnancy test. 1 genetic change, 1 positive filipin stain, and a positive • In the case of women of child-bearing potential, commit to biomarker. use highly effective contraceptive measures, which include • Have mild to moderate cognitive impairment with a global barrier methods in combination with other contraceptive Clinical Dementia Rating (CDR) score of 0.5 to 2 and/or a measures (e.g., oral, or other hormonal contraceptives) or CDR-SoB ≤ 12. commit to sexual abstinence until at least 12 weeks after • Have the capacity (at the investigator’s discretion) to the final dose of EFV. understand the nature and risks of the trial and the rele- • In the case of male patients, commit to use barrier contra- vant information given, the capacity to decide whether to ceptives (condoms) until at least 12 weeks after the final participate, and sign the written informed consent. dose of EFV. • Have a live-in caregiver available more than 5 days a week • Sign the written informed consent. that can inform the patient’s medical situation, under- stand the nature and risks of the trial, and sign the written Note: Although the primary target population of the study informed consent. is patients with adult-onset NPC (i.e., patients > 16 years old), • Be proficient in Spanish (the language in which cognitive we will also enroll patients aged between 14 and 16 years old tests will be conducted). (patients with late juvenile-onset NPC). The justification for • Have sufficient visual and auditory acuity for understand- including the late juvenile population (between 14 and 16 years ing explanations given and neuropsychological tests. old) in the study is that NPC also causes cognitive impairment in • Have a minimum 8 years of schooling. this age interval. 3 Gascón-Bayarri et al. • Medicine (2022) 101:48Medicine 2.2.2. Exclusion criteria. Despite this established 25 mg/d dose, successive blood analy- ses will be performed during the trial, and those patients with • Any sign or suspicion of a diagnosis other than NPC. no evidence of a blood 24-OH-Cholesterol increase ≥ 1.5 times • Active Central Nervous System infections that may inter- their baseline levels will have their EFV dose increased up to fere with normal brain function. 200 mg/d. • History of severe traumatic head injury. According to the SmPC, the recommended antiretroviral dose • History of severe psychiatric illnesses: bipolar disorder, of EFV for adults is 600 mg/d PO, combined with nucleoside schizophrenia, major depressive disorder. reverse transcriptase antiretroviral inhibitor with or without a • Risk of suicide, according to the live-in caregiver or investigator. protease inhibitor. The dose selected for this trial (25 mg/d) is • History of substance abuse or dependence. the 1 administered to the pediatric population with 3.5 to <5 kg • Presence of cerebrovascular disease that can explain the of weight. Also, as recommended by the SmPC, patients will be cognitive impairment (in the investigator’s opinion). advised to take the medication at bedtime. • Inability to tolerate the study-related procedures. • Stage IV chronic kidney disease. 2.3.1. Rationale for dose selection. Preclinical studies using • Chronic liver disease or increased Aspartate NPC mouse models used an Efavirenz dose 300 times smaller Aminotransferase or Alanine Aminotransferase levels (≥3 than the 1 used in HIV patients.[14] Although very small, the times the upper limits of normal). dose used has been proven to activate CYP46 and efficiently • Chronic viral hepatitis B or C. promoted the recycling of cholesterol in the brains of normal • Patients with systemic immunosuppression. mice.[14] • Unstable epilepsy (at the investigator’s discretion) in the However, extrapolating preclinical doses to a clinical setting last 3 months*. is challenging. The significant differences in body size, brain size, and the blood-brain barrier must be considered, among other *Note: Those with controlled/stable epilepsy can be enrolled. things. Also, administering humans with a dose as low as the 1 • History of malignancies not considered stable or cured (in administered in mice would risk not having the desired effect. general, patients who have not been treatment-free for at Notably, 1 clinical trial (NCT03706885, ClinicalTrials.gov) is least 5 years before screening and considered to be at low currently being conducted in patients with Alzheimer’s dementia risk of recurrence by the treating physician). using oral doses of EFV of 50 and 200 mg/d to activate CYP46 • Severe dementia that hinders the completion of cognitive in the brain. tests and evaluations. In light of all that, the study team has selected a dose of • History of allergy/hypersensitivity to EFV or to any of the 25 mg/d of EFV, considering that the desired effect should be excipients found in the formula. observed with this dose – a dose 24 times smaller than the recom- • Patients using anticoagulants are not excluded from the mended antiretroviral dose for HIV patients (600 mg/d). Given trial but will NOT be offered the possibility to join the that increases in plasma 24-OH-Cholesterol concentrations lumbar puncture group. (≥1.5 times the pre-EFV baseline values) have been observed in • Concomitant treatment with any of the following: ter- the preclinical studies, we will perform successive blood tests fenadine, astemizole, cisapride, midazolam, triazolam, (every 3 months). If no 24-OH-Cholesterol increases ≥ 1.5 times pimozide, bepridil, or ergot alkaloids (e.g., ergotamine, the pre-EFV baseline values are observed, we shall consider dihydroergotamine, ergonovine, and methylergonovine)*. increasing the dose to up to 200 mg/d. *Note: These drugs are substrates of the CYP3A4 that could cause a competitive inhibition of the EFV metabolism and gen- 2.4. Criteria for discontinuing or modifying allocated erate serious and/or life-threatening AEs (e.g., cardiac arrhyth- interventions mias, prolonged sedation, respiratory depression). As mentioned before, all enrolled patients will receive EFV + SoC. • Co-administration with elbasvir and grazoprevir due to the The EFV will be discontinued if the investigator deems that it potential of EFV to significantly decrease plasma concen- poses an unacceptable risk to the patient; the patient withdraws trations of both drugs. his consent; there is a significant violation of the study proto- • Use of herbal medicinal products containing St. John’s col; there is a loss to follow-up; or in case a study participant Wort (Hypericum perforatum) due to the risk of decreasing becomes pregnant. the plasma concentrations and the clinical effects of EFV. • Family history of sudden death or congenital prolongation of the QTc interval or any other clinical condition known 2.5. Strategies to improve adherence to intervention to prolong the QTc interval. In addition to collecting and reviewing empty medication boxes, • Family history of symptomatic cardiac arrhythmias, clini- patients will be provided with a “Patient’s diary” for better con- cally relevant bradycardia, or congestive heart failure asso- trolling treatment adherence. ciated with a reduced left ventricular ejection fraction. • Severe disturbances of electrolyte balance, such as hypoka- lemia or hypomagnesemia. 2.6. Relevant concomitant care permitted or prohibited • Concomitant treatment with drugs known to prolong the during the trial QTc interval, such as class IA and class III antiarrhyth- mics; neuroleptics; antidepressant drugs; certain antibiotics As mentioned in the exclusion criteria, the following concomi- (i.e., macrolides, fluoroquinolones); imidazole and triazole tant medications are prohibited during the trial: antifungal agents; certain non-sedating antihistamines (i.e., • CYP3A4 inhibitors, including, but not limited to ter- terfenadine, astemizole); cisapride; flecainide; certain anti- fenadine, astemizole, cisapride, midazolam, triazolam, malarials; and methadone. pimozide, bepridil, or ergot alkaloids (e.g., ergotamine, dihydroergotamine, ergonovine, and methylergonovine). Substrates that compete with EFV for CYP3A4-binding 2.3. Interventions could decrease its metabolism and induce serious and/or The study intervention is EFV. All patients will be treated orally life-threatening adverse reactions (e.g., arrhythmias, pro- with 25 mg/d of EFV for 52 weeks (1 year), in addition to SoC. longed sedation, or respiratory depression). 4 Gascón-Bayarri et al. • Medicine (2022) 101:48www.md-journal.com • Elbasvir and Grazoprevir, because EFV has the potential to (terfenadine, astemizole); cisapride; flecainide; certain anti- significantly decrease plasma concentrations of both drugs. malarials; and methadone. • Herbal medicinal products containing St. John’s Wort (Hypericum perforatum) due to the risk of decreasing plasma concentrations and the clinical effects of EFV. 2.7. Outcomes: • Drugs known to prolong the QTc interval, including, but not limited to class IA and class III antiarrhythmics; neu- Primary and secondary outcomes are summarized in Table 2. roleptics; antidepressant agents; certain antibiotics (e.g., A deterioration in the CDR-SoB is defined as an increase macrolides, fluoroquinolones, and imidazole and triazole of ≥ 2 points at 52 weeks of starting treatment with EFV rela- antifungal agents); certain non-sedating antihistamines tive to baseline. Table 2 Summarized outcome measures. Primary outcome measure Change (not deterioration) in cognitive performance at 52 wks of starting treatment with EFV, assessed by:  CDR-SoB score.  FCSRT score (to evaluate verbal memory).  The executive functions as per the following: a)a-BT scores in the digit span subtest. b)a-BT scores in the mental control subtest. c)Scores in verbal fluency subtest: semantic and phonemic tasks. d)TMT A and B scores e)SCWT scores. Secondary outcome measures Clinical Neurological: Changes at 52 wks of starting treatment with EFV relative to baseline in:  SARA score  EAT-10 score.  PDS score • Neuropsychological (protocol used in clinical practice): In general, the scaled score will be determined for the following questionnaires/scales:  CDR-SoB* score. *Note: It offers a specific score from 0 to 18, the higher the score the greater the severity.  FCSRT score  a-BT assessment in the following domains: ◦Orientation, digit span, automatized sequences (e.g., counting to 20, d of the wk, mo of the yr), comprehension of verbal commands, and comprehension of complex verbal material (i.e., yes-or-no answers to BDAE-based questions) subtests’ scores. ◦Bilateral gesture-based limb praxis test ◦Visual memory (i.e., figures) ◦Reading, repeating, and writing ◦Mental and written arithmetic ◦Reciprocal coordination ◦Unilateral alternating sequences subtest (e.g., serial hand tests, such as fist-palm-side, tapping). ◦Constructional praxis (drawing copy).  Verbal Fluency score: Semantic and phonemic tasks  TMT A and B scores  SCWT score  WAIS-III/WAIS IV  BNT, Naming and Semantic Knowledge subtests’ scores  JLO Test score.  DEX questionnaire score.  NPI score.  AES score.  BDI score.  C-SSRS score. Biological • Brain 18FDG PET–Scan: Quantification of cerebellar, cortical, and thalamic metabolism. • Brain MRI: Cortical, white matter, basal ganglia, and cerebellar structures volumetric analysis. • Abdominal ultrasound: Spleen size (cm). • Oculography: initial speed, amplitude, and execution speed of vertical and horizontal saccades • Plasma Oxysterols levels • Plasma Lyso-SM-509 level • Plasma 24-OH-Cholesterol level • Cerebrospinal Fluid (CSF) Beta-Amyloid, Tau, and phosphorylated Tau levels Safety • Number of adverse events (AEs) according to seriousness, severity, and relationship with EFV. • Changes in the liver function panel 8 FDG PET—scan = 18Fluorodeoxyglucose Positron emission tomography scan, a-BT = abbreviated Barcelona test, AES = apathy evaluation scale, BDI = Beck’s depression inventory, BNT = Boston naming test, CDR-SoB = clinical dementia rating scale-sum of boxes, C-SSRS = Columbia suicide severity rating scale, DEX = dysexecutive questionnaire, EAT-10 = the eating assessment tool score, EFV = efavirenz, FCSRT = free and cued selective reminding test, JLO = judgement of line orientation test, MRI = magnetic resonance imaging, NPC = Niemann-Pick disease type C, NPI = neuropsychiatric inventory, PDS = Pineda disability scale, SARA = scale for the assessment and rating of ataxia, STROOP = Stroop color and word test, TMT = trail making test, WAIS = Wechsler adult intelligence scale. 5 Gascón-Bayarri et al. • Medicine (2022) 101:48Medicine A deterioration in verbal memory is defined as a decrease supplied EFV is to be used exclusively in this clinical trial. The of ≥ 1 in the standard deviation (SD) at 52 weeks of starting Pharmacy Department will have an updated record of the EFV treatment with EFV relative to baseline. and manage an inventory that will collect all product entries, A deterioration in the executive function is defined as dispensing, returns, and disposals. a decrease of ≥ 1 in the SD (in at least 2 of the 5 tests per- The records will comply with current regulations and guidelines. formed) at 52 weeks of starting treatment with EFV relative Once they are completed, they must be signed and dated by the to baseline. person in charge. The clinical trial pharmacist will be responsible A patient will be considered a responder (no decay in cog- for maintaining all records of the medication used. The EFV does nitive performance) if there is no deterioration in at least 2 of not require any special storage conditions according to the SmPC the 3 assessed items at 52 weeks of starting treatment with EFV and should not be used after the expiry date indicated on the label. relative to baseline. It is the investigator’s responsibility to ensure that the necessary measures have been taken for proper EFV disposal, as required 2.7.1. Study procedure. Study candidates will be screened by applicable legislation, guidelines, and institutional procedures. during visit 1. Those who meet all the inclusion criteria and none of the exclusion criteria will be invited to participate. Both patient and caregiver must sign the written informed consent, a 2.8. Sample size sine qua non condition for enrollment. Seven study visits will be performed for enrolled patients, and all the information detailed We have not performed a formal sample size calculation. Adult in Table 3 will be gathered. NPC is an orphan disease, with an estimated prevalence of 27 We estimate it will take approximately 3 months to complete patients in Spain in 2019. The principal investigator (PI) cur- the enrollment procedure. We expect to carry out the study rently follows 14 of these patients at the BUH. Therefore, we between 2022 and 2024. expect to enroll a total of 14 patients. By the moment of submitting this manuscript, participant recruitment (patients’ screening visit) was still in progress. Participant Timeline 2.9. Recruitment The EFV will be provided by the study sponsor and will The study patients will be recruited from the BUH Neurology be acquired through the Pharmacy Department at BUH. The Department. The PI will recruit patients chronologically, as they Table 3 Participant timeline. Visit 1 (baseline visit) Visit 2 Visit 2.1 Visit 3 Visit 4 Visit 5 Visit 6 Screening and Start of End of enrollment treatment treatment Day 0 Wk 4 ± 3 d Wk 13 ± 1 wk Wk 26 ± 1 wk Wk 39 ± 1 wk Wk 52 ± 2 wks −60/−30 d (Wk 0) after visit 2 after visit 2 after visit 2 after visit 2 after visit 2 Inclusion/exclusion criteria ✓ ✓ Written informed consent ✓ Demographic data* ✓ Vital signs (BP, HR, T, RR) and body weight ✓ ✓ ✓ ✓ ✓ ✓ ✓ Physical examination ✓ ✓ ✓ ✓ ✓ ✓ ✓ Neurological examination‡ ✓ ✓ ✓ ✓ ✓ ✓ ✓ Neuropsychological assessment§ ✓ ✓ ✓ General blood tests‖ ✓ ✓ ✓ ✓ ✓ Biomarkers:  24-OH-cholesterol ✓ ✓ ✓ ✓ ✓ Biomarkers: Oxysterols  Lyso-sm-509 ✓ ✓ ✓ EKG ✓ ✓ ✓ ✓ ✓ Medication dispensing ✓ ✓ ✓ ✓ ✓ Brain MRI ✓ ✓ Brain 18FDG-PET scan ✓ ✓ ✓ Oculography ✓ ✓ ✓ Abdominal ultrasound ✓ ✓ ✓ Lumbar puncture ✓† ✓† AE recording ✓ ✓ ✓ ✓ ✓ ✓ ✓ Concomitant medication recording ✓ ✓ ✓ ✓ ✓ ✓ ✓ Patient’s journal ✓ ✓ ✓ ✓ ✓ Pregnancy test ✓ ✓ ✓ ✓ ✓ ✓ AE = adverse event, BP = blood pressure, EKG = electrocardiogram, HR = heart rate, MRI = magnetic resonance imaging, RR = respiratory rate, T = temperature. (1) Includes genetic diagnosis. ‡Includes Ataxia Scale (SARA), Dysphagia Scale (EAT-10), Pineda Disability Scale. §CDR-SoB: Clinical Dementia Rating-Sum of Boxes Scale; a-BT: Abbreviated Barcelona Test; WAIS-III/WAIS-IV: Weschler Adult Intelligence Scale, Cubes, Symbol Search, and Vocabulary subtests; FCSRT: Free and Cued Selective Reminding Test; Verbal Fluency Score: Semantic and phonemic tasks (category fluency [CF] and letter fluency [LF]); TMT: Trail Making Test A and B; SCWD: Stroop Color and Word Test; BNT: Boston Test, subtests Naming and Semantic Knowledge Test; JLO: Judgement of Line Orientation Test; DEX: Dysexecution Questionnaire; NPI: Neuropsychiatric Inventory; AES: Apathy Evaluation Scale; BDI: Beck Depression Scale; C-SSRS: Columbia Suicide Severity Risk Scale. ‖Complete blood count, coagulation tests, and Biochemistry (sodium, potassium, calcium, protein, glucose, urea, creatinine, aspartate aminotransferase, alanine aminotransferase, cholesterol, TSH, T4). †Lumbar puncture will be performed to determine CSF biomarkers (i.e., Beta-amyloid, Tau, and phosphorylated Tau) in those patients who agree to undergo this procedure. The investigator must inform patients and caregivers that this procedure is optional and about the risks involved. Patients who accept it will be required to sign a separate and specific written informed consent (both patients and caregivers must sign it). 6 Gascón-Bayarri et al. • Medicine (2022) 101:48www.md-journal.com come to the BUH, when they meet the selection criteria specified 2.15. Methods for any additional analyses before, until reaching the 14 expected patients. A descriptive analysis of the AEs recorded throughout the trial will be carried out for patients enrolled in the safety analysis set. 2.10. Assignment of interventions 2.10.1. Allocation: Sequence generation, concealment 2.16. Analysis population and missing data definition of mechanism, implementation, and blinding (Masking). Not analysis population relating to protocol nonadherence and applicable. This is a single-arm clinical trial; thus, all enrolled any statistical methods to handle missing data patients will receive the study treatment. In case of missing data, imputation will be made considering the treatment effects’ estimator is not biased and that an increase in type I error has been avoided. However, assuming that the 2.11. Data collection plan cognitive impairment will slow down and aiming to ensure the Data collection will be performed during the study visits from both trial’s internal sensitivity (in case of expecting a different evo- patient interviews and electronic medical registers and will be intro- lution of one of the experimental groups), the last observation duced in an e-CRF created ad hoc. A data curation process will be carried forward technique seems to be a conservative approach performed to improve data quality and reduce possible errors. to the matter. 2.12. Plans to promote participant retention and complete 2.17. Oversight and monitoring follow-up 2.17.1. Composition of the data monitoring committee, BUH is the reference center of the Spanish National Health its role and reporting structure. A Data Safety Monitoring System for NPC patients at a national level, and the PI (JG) has Committee will be established for this trial and will consist of been the person in charge of managing these patients for the last members of the study team and third-party pharmacovigilance years. Therefore, the doctor-patient relationship is very close. experts (from the BUH). The data safety monitoring committee Also, to date, only 27 patients have been diagnosed with NPC in will evaluate all the available EFV safety data at each study visit Spain. Altogether, this makes it easier to retain the participants performed during the trial. and complete the follow-up procedure. During the development of the study, the sponsor will prepare Drug Safety Update Reports (DSURs) annually, following the 2.13. Data management recommendations of the ICH E2F guideline, and present them to the regulatory authorities and the IRB following the calendar An electronic case report form (e-CRF) will be created ad-hoc for established in the current legislation. this study based on Research Electronic Data Capture Consortium The sponsor will inform the investigators of any new safety platform. It will not collect data that allows patient identification. information that could jeopardize the patients’ safety as soon as Comprehensive data will be collected, including the date of possible. The investigators will also be notified of any modifica- patient enrollment, the date of the first administration of EFV, tion to the study protocol. demographic data, medical history, relevant comorbidities, clin- ical data, any concomitant medications administered, and the results of the biochemical analyses mentioned before. 2.18. Description of any interim analyses and stopping The study monitoring will be carried out by a Contract guideline Research Organization (OPTIMAPHARM, https://optima- pharm.eu/; accessed on August 1st, 2022). No interim analyses have been planned for this trial. 2.14. Statistical methods for analyzing primary and 2.19. Adverse event reporting and harms secondary outcomes Given that EFV is an authorized medicinal product, the SmPC We will perform a descriptive analysis of the enrolled patients’ will be used as the Reference Safety Information. baseline sociodemographic and clinical data as a whole and by The investigators will systematically monitor and collect study groups. the AEs from the moment of signing the written informed The primary outcome measure will be evaluated by an consent to the patient’s last follow-up visit. AEs will be analysis of covariance (ANCOVA). Cognitive performance recorded in the patient’s medical record, stating the causality (assessed by the previously described neuropsychological test assessment for EFV. AEs related to the EFV will be collected battery at 52 weeks) will be the dependent variable, and the through the eCRF whenever they are serious (SAEs) or AEs of independent variables will be the study group and the cog- special interest. nitive performance assessed by the CDR-SoB at the baseline The investigators must provide a thorough description of visit. Tables will be presented with model coefficients and the event, including start and end dates, an intensity assessment their 95% confidence intervals (95% CI). Additionally, the (mild, moderate, or severe), measures taken to mitigate it (e.g., effect size will be measured by presenting the marginal means none, EFV withdrawal, the medication used to treat the AE), per study group and their standardized difference along with and a causality assessment for EFV. their 95% CI. Any AE or clinically relevant lab abnormality will be fol- Given the exploratory nature of the clinical and biologi- lowed until satisfactory resolution, stabilization, or until clinical cal secondary outcome measures, we will use the same sta- judgment indicates it has an alternative cause and no further tistical technique outlined for the primary outcome measure. assessment is needed. However, either generalized models or non-parametric statis- The investigator must notify the contracted contract research tics will be used depending on the distribution of the depen- organization of any SAE (including death from any cause) in dent variable. less than 24 hour after becoming aware of it, regardless of its R version 3.6 or higher will be used to perform the statistical relationship with EFV. This will be done by sending a specific analysis. notification form by fax or email. 7 Gascón-Bayarri et al. • Medicine (2022) 101:48Medicine Any suspected unexpected serious adverse reaction (i.e., those 2.24. Additional consent provisions for collection and use not reported in the Reference Safety Information) will be of of participant data and biological specimens expedited reporting as per the current Spanish (Royal Decree 1090/2015) and European legislation (Regulation EU 536/2014). As mentioned before, an optional lumbar puncture will be For the purpose of this trial, we consider as AEs of special performed to determine CSF biomarkers (i.e., Beta-amyloid, interests laboratory abnormalities in plasma liver enzymes Tau, and phosphorylated Tau) in those patients who agree to (aspartate aminotransferase and alanine aminotransferase > 2× undergo this procedure. An additional written informed consent upper limits of normal). is required prior to performing the lumbar puncture. Samples In the event of a pregnancy during the study, the EFV will will be stored and patients will be informed about the storage be immediately withdrawn. The investigator must notify the along with all study procedures by the recruiting investigator sponsor within 24 hours of becoming aware of the event. (see item 3.3). Appropriate medical assistance will be offered to the patient. These patients will be asked to sign a specific written informed 2.25. Confidentiality consent allowing the investigators to collect pregnancy-related data. The patient will be followed until the delivery date; if the The results from this clinical trial are confidential and may not outcome meets SAE criteria, the investigators will follow the be transferred to third parties in any form or manner without procedures explained before for SAE notification. written permission from the Sponsor. All individuals involved in the clinical trial are bound to this confidentiality clause in line with the Regulation (EU) 2016/679 of the European Parliament 2.20. Frequency and procedures for auditing trial conduct and of the Council of April 27th, 2016, on the protection of nat- The Investigator shall allow direct access to trial data and doc- ural persons concerning the processing of personal data and the uments for monitoring, audits and/or inspections by competent free movement of such data, as well as all other valid and appli- regulatory or health authorities. As such, e-CRFs, source records cable laws and regulations, such as the “Organic Law 3/2018 and other trial-related documentation must be kept current, on Personal Data Protection and Digital Rights Assurance. complete, and accurate at all times. Therefore, patient data will be anonymized. While obtaining a signature for the Written Informed Consent, the investigator will request written permission from 2.21. Ethics approval and consent to participate the patient to directly access his/her data. With this permission The study protocol version 3.0 (February 10th, 2020), EudraCT granted, the patient’s data may be examined, analyzed, verified, number 2019-004498-18, received local IRB approval (Ethics and reproduced for the clinical trial evaluation. and Clinical Investigation Committee of the Bellvitge University Data will be anonymized so that the corresponding patient Hospital, code AC049/19) on February 27th, 2020). Notably, cannot be identified. Patient data will also be dissociated. Patients due to the COVID-19 pandemic and the difficulty obtaining will be assigned consecutive numbers as they are enrolled in the the study medication (EFV), the start of this clinical trial was study, and these identification numbers (or codes) will be used halted for 2 years. After this period, the protocol was updated in the e-CRF; the patient’s full name will not be included in to version 3.1 (March 24th, 2020). The local IRB approved this the e-CRFs. The principal investigator of each center will keep new version on April 15th, 2022. The list of local IRB members an updated patient identification list containing the name, clin- is available at https://bellvitgehospital.cat/es/investiga-con-no- ical history number, and the patient’s identification number (or sotros/ceic/composicion. Accessed on August 1st, 2022. code) for the clinical trial. This Trial will be conducted according to the criteria set by The study monitor may have access to the patient’s identity the Declaration of Helsinki (revised on WMA 64th General and data related to the study monitoring procedures. Any per- Assembly, Fortaleza, Brazil, October 2013), good clinical prac- son with direct access to the data (Regulatory Authorities, Trial tice standards and applicable regulations. A specific insurance Monitors, and auditors) will take all possible precautions to will be hired for this clinical trial. maintain the confidentiality of patients’ identities. The level of confidentiality protection, in terms of personal It is the investigator’s responsibility to obtain written informed data protection, as required by Spanish (Organic Law on consent from the study patients. It is the Trial Monitor’s respon- Data Protection 3/2018) and European Law (Regulation EU sibility to make sure that each patient has given his/her written 2016/679 of the European Parliament and Council), was also consent to allow this direct access. ensured (see item 2.25). The investigator shall ensure that the documents provided to Every patient that accepts to participate in the study will be the Sponsor do not contain the patient’s name or any identifi- assigned consecutive numbers as they are enrolled, and these able data. numbers (or codes) will be used in the eCRF, instead of personal Dr Cristian Tebé, Head of the Biostatistics Unit, will oversee data. The data collected will be encoded, so that the patient to the final trial dataset, along with the PI. whom they correspond is not identified. 2.26. Ancillary and post-trial care 2.22. Plans for communicating important protocol As mentioned before, BUH is the reference center of the National amendments to relevant parties Health System for NPC patients at a national level, and the PI Major protocol changes will be submitted for IRB approval and (JG) has been and still is the person in charge of managing these minor outcomes will be informed to the IRB. As per good clini- patients during the last years. Therefore, the PI will remain to cal practice, trial participants will be informed of any significant be these patients’ assistant physician after the trial ends. On the changes during the trial. other hand, a specific insurance has been hired ad hoc in case of any harm related to a patient’s participation. 2.23. Who will obtain informed consent? The PI (or another physician from the study team) will inform 2.27. Dissemination policy: trial results, authorship the screened patients about the study and ask them to sign the The study findings will be submitted to a peer-reviewed journal written informed consent in visit 1 if the patient is interested in for publication and presented at relevant national and interna- participating in the study. tional scientific meetings. 8 Gascón-Bayarri et al. • Medicine (2022) 101:48www.md-journal.com The authorship is based on the criteria established by the 3.2. Informed consent materials International Committee of Medical Journal Editors (https:// Please refer to Supplementary Material, http://links.lww.com/ www.icmje.org/recommendations/browse/roles-andrespon- MD/I123. sibilities/defining-the-role-of-authors-and-contributors.html, accessed on August 1st, 2022). 3.3. Plans for collection, laboratory evaluation, and storage of 2.28. Plans to give access to the full protocol, participant biological specimens for genetic or molecular analysis in the level-data and statistical code current trial and for future use in ancillary studies, if applicable The protocol is available on EudraCT Number: 2019-004498- Biological samples from blood tests and CSF analysis will be 18. No public access to the patient dataset is planned to be given analyzed during the conduct of the trial and will be stored in at this moment. Dr Cristian Tebé, the Head of the Biostatistics the HUB-ICO-IDIBELL Biobank, which manages and regu- Unit, will oversee the dataset and granting access to this infor- lates the different biological samples collected at the BUH, mation will be evaluated on a case-by-case basis and upon rea- Catalan Institute of Oncology (ICO), and other associated sonable request by the interested part. centers, under the current Spanish law 14/2007 and the Royal Decree 1716/2011. Additional information on the HUB-ICO- IDIBELL Biobank can be found at https://idibell.cat/en/services/ scientific-and-technical-services/biobank/. 3. Discussion Patients will be informed about the biological samples’ stor- NPC is a complex orphan disease, consisting of an unmet med- age along with all study procedures by the recruiting investi- ical need for patients suffering from it. Although there are over gator. This information will also be displayed in the Patient 20 ongoing studies on NPC registered in the ClinicalTrials. Information Sheet, and all patients must sign the written gov database, conducting clinical trials for assessing new ther- informed consent prior to any study-related procedure, includ- apeutic options can be quite challenging, given the disease’s ing the storage of any biological sample. low prevalence. Different therapeutic approaches are under study (e.g., Miglustat, Arimoclomol, N-Acetyl-L-Leucine), Acknowledgments but, to the best of our knowledge, this is the first clinical trial studying the effects of EFV in adult- and late-juvenile-onset We thank the Spanish Niemann-Pick Foundation for provid- NPC. ing financial support. We also thank the Bellvitge University As mentioned before, neuronal cholesterol metabolism is Hospital, IDIBELL and CERCA Program/Generalitat de key for LTP, which is a major event for synaptic plasticity. The Catalunya for institutional support. enzyme CYP46A1 hydroxylates the cholesterol and mediates the main pathway for its degradation in the brain.[9,10] Given Author contributions that NPC1 binds cholesterol and participates in its intracel- lular mobilization, we hypothesized that NPC1 would have a Conceptualization: Jordi Gascón-Bayarri, Petru Cristian Simon, synapse-specific role in redistributing cholesterol and mobiliz- Sebastián Videla. ing CYP46A1 to the synaptic membrane required for LTP. We Funding acquisition: Jordi Gascón-Bayarri, María Dolores believe that the capacity of EFV to activate the CYP46A1 could Ledesma, Sebastián Videla. compensate for NPC1 deficiency and correct synaptic changes, Investigation: Jordi Gascón-Bayarri, Imma Rico, Cristina therefore compensating cognitive and psychiatric alterations in Sánchez-Castañeda, Jaume Campdelacreu-Fumadó, Nahum patients with NPC. This hypothesis is supported by preclinical Calvo-Malvar, Mònica Cos, Eugenia de Lama, Montserrat data[12,14] suggesting that low-dose EFV may be a promising Cortés-Romera, Laura Rodríguez-Bel, Celia Pérez-Sousa, alternative for NPC patients. María Cerdán Sánchez, Nuria Muelas, María Dolores On the other hand, this study has limitations. First, the already Sevillano, Pablo Mir, Adolfo López de Munain, Anna Ferrer. mentioned small sample size due to the disease’s low prevalence. Methodology: Jordi Gascón-Bayarri, Petru Cristian Simon, Therefore, interpreting the results may be tough, given that we Thiago Carnaval, Sebastián Videla. cannot safely exclude that a given outcome happened by chance. Supervision: Jordi Gascón-Bayarri, Roser Llop. Second, it has a single-arm design (absence of a control group). Writing – original draft: Jordi Gascón-Bayarri, Thiago Carnaval, Although justifiable by the natural history of the disease, the Sebastián Videla. unexpectedness of spontaneous improvement, and the lack of Writing – review & editing: Jordi Gascón-Bayarri, Petru Cristian expected placebo effects, this type of design has idiosyncratic Simon, Thiago Carnaval, María Dolores Ledesma, Sebastián limitations since it could be argued that responders would have Videla. responded equally if treated only with SoC. Furthermore, the absence of a frame of reference for comparison could also make References the results hard to interpret. This study may provide direct benefit to enrolled patients in [1] Brady RO, Kanfer JN, Mock MB, et al. The metabolism of sphingomy- terms of slowing down the disease progression. If the preclin- elin. II. Evidence of an enzymatic deficiency in Niemann-Pick disease. Proc Natl Acad Sci USA. 1966;55:366–9. ical results are reproducible in a clinical setting, NPC patients [2] Higgins ME, Davies JP, Chen FW, et al. Niemann-Pick C1 is a late endo- may have a new therapeutic option in the future to address some-resident protein that transiently associates with lysosomes and their currently unmet medical needs. Nonetheless, future Phase the trans-Golgi network. Mol Genet Metab. 1999;68:1–13. III clinical trials would be needed to confirm the findings of [3] Watari H, Blanchette-Mackie EJ, Dwyer NK, et al. Niemann-Pick C1 this trial. protein: obligatory roles for N-terminal domains and lysosomal targeting *Note: This protocol was written following the SPIRIT in cholesterol mobilization. Proc Natl Acad Sci USA. 1999;96:805–10. statement.[15] [4] Vance JE, Karten B. Niemann-Pick C disease and mobilization of lyso- somal cholesterol by cyclodextrin. J Lipid Res. 2014;55:1609–21. [5] Parton RG, Simons K, Dotti CG. Axonal and dendritic endocytic path- ways in cultured neurons. J Cell Biol. 1992;119:123–37. 3.1. Trial status [6] Karten B, Vance DE, Campenot RB, et al. Trafficking of cholesterol The current protocol version is 3.1 (March 24th, 2022). This from cell bodies to distal axons in Niemann Pick C1-deficient neurons. trial is currently on the screening phase (August 2022). J Biol Chem. 2003;278:4168–75. 9 Gascón-Bayarri et al. • Medicine (2022) 101:48Medicine [7] Karten B, Campenot RB, Vance DE, et al. The Niemann-Pick C1 pro- comparable to commonly observed human mutations. Hum Mol tein in recycling endosomes of presynaptic nerve terminals. J Lipid Res. Genet. 2012;21:730–50. 2006;47:504–14. [12] Mitroi DN, Pereyra-Gomez G, Soto-Huelin B, et al. NPC1 enables cho- [8] Brachet A, Norwood S, Brouwers JF, et al. LTP-triggered cholesterol lesterol mobilization during long-term potentiation that can be restored redistribution activates Cdc42 and drives AMPA receptor synaptic in Niemann-Pick disease type C by CYP46A1 activation. EMBO Rep. delivery. J Cell Biol. 2015;208:791–806. 2019;20:e48143. [9] Sodero AO, Weissmann C, Ledesma MD, et al. Cellular stress from [13] De Clercq E. Anti-HIV drugs: 25 compounds approved within 25 years excitatory neurotransmission contributes to cholesterol loss in hippo- after the discovery of HIV. Int J Antimicrob Agents. 2009;33:307–20. campal neurons aging in vitro. Neurobiol Aging. 2011;32:1043–53. [14] Mast N, Li Y, Linger M, et al. Pharmacologic stimulation of cyto- [10] Sodero AO, Vriens J, Ghosh D, et al. Cholesterol loss during gluta- chrome P450 46A1 and cerebral cholesterol turnover in mice. J Biol mate-mediated excitotoxicity. EMBO J. 2012;31:1764–73. Chem. 2014;289:3529–38. [11] Maue RA, Burgess RW, Wang B, et al. A novel mouse model of [15] Chan AW, Tetzlaff JM, Altman DG, et al. SPIRIT 2013 statement: defining Niemann-Pick type C disease carrying a D1005G-Npc1 mutation standard protocol items for clinical trials. Ann Intern Med. 2013;158:200–7. 10